Isoprenoid compounds comprise the largest family of small molecules in nature. Many are essential for cells to survive and elements of the isoprenoid biosynthetic pathway are found in all organisms. Several isoprenoid biosynthetic enzymes are targets for drugs. Lipitor, the most widely prescribed drug, targets an enzyme early in the pathway to lower serum cholesterol. Fosamax, used to treat osteoporosis, targets a chain elongation enzyme in the middle of the pathway. The antifungal drug Lamisil targets the later stages of fungal steroid biosynthesis. Differences between the isoprenoid biosynthetic pathways and enzymes in humans and microorganisms present new attractive targets for drug development. In addition, products of the pathway have a long history as drugs, including more recent examples such as taxol (cancer) and artemisinin (malaria). This project supports research to elucidate new biosynthetic routes to isoprenoid compounds and structural and mechanistic studies of enzymes in the pathways from where the routes diverge from fatty acid metabolism and glycolysis up to the synthesis of the two fundamental five-carbon building blocks of isoprenoid molecules, isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP). The work incorporates a combination of genetics, molecular biology, mechanistic enzymology, structural biology, and synthetic organic chemistry in this effort. The PI's laboratory is a unique site for this project. He and his coworkers have expertise specifically related to the isoprenoid pathway in the diverse areas of research outlined above. His laboratory has a large collection of bacterial and fungal clones bearing knockouts or additions of appropriate biosynthetic enzymes, expression vectors for production of purified enzymes, substrates/alternate substrates/inhibitors, and the analytical tools required for identification of new metabolites and for structural and mechanistic studies. Three interrelated areas of research are proposed for the coming grant period. (1) All available evidence indicates that most, if not all, Archaea synthesize isopentenyl diphosphate from mevalonate differently that eukaryotes. Specifically, mevalonate phosphate is first converted to isopentenyl phosphate (IP) and then to the diphosphate. The gene for synthesis of IP is missing. Identification of the gene and verification of its function will be undertaken. Related work on X-ray structure-function and mutagenesis studies of IP kinase will be conducted, including the development of mutants capable of efficiently synthesizing longer chain diphosphates from the corresponding phosphates. (2) Isoforms of IPP isomerase (IDI1 and IDI2) convert IPP to DMAPP by a proton-initiated process. NMR, X-ray, UV, and fluorescence experiments will be conducted to identify the amino acids/cofactors involved in the proton transfer reactions, including the unusual role of reduced flavin in the proton transfer reactions of IDI2. (3) Amine and thiol-containing analogues of methylerythritol phosphate and hydroxydimethylallyl diphosphate will be synthesized and examined as mechanism-based inhibitors of the iron-sulfur cluster enzymes the methylerythritol phosphate pathway.